Primary prevention of breast cancer requires using models of human non-neoplastic cells to decipher the mechanisms of tumor initiation and to design much needed preventive strategies. The HMT3522 S1 non- neoplastic cells mimic the physiologically relevant breast epithelial development by forming ball-shaped three- dimensional (3D) polarized (basal/apical) glandular units (acini) when cultured in contact with an appropriate extracellular matrix (3D culture). Using this model we have identified the loss of apical polarity as a necessary event for cell cycle entry. This suggests that apical polarity is likely to be a ver early architectural modification reflecting an increased risk for tumor onset. Environmental and genetic/epigenetic risk factors for breast cancer have been seldom studied in relevant cell culture models. An important direction of research is to develop cell lines from women with heightened breast cancer risk to unravel the architectural and epigenetic determinants of breast tumor development. Therefore, two research teams for this project are sharing their complementary expertise in the phenotypical analysis of cells in 3D culture and the development and genomic analysis of lines of non-neoplastic cells from women at low or high breast cancer risk. Our goal is to develop high-throughput human cell-based models for the screening of markers of risk assessment and preventive agents. The innovative idea is to use cell lines that represent different breast cancer risks to identify epigenetic markers for elevated risk by correlating these markers to a weakened acinar architecture (i.e., apical polarity loss). The rationale for focusing on epigenetic markers, principally histone modifications as a proof of principle, is that epigenetics is at the heart of gene expression control that goes awry in cancer. Moreover, epigenetic mechanisms are strongly influenced by breast cancer risk factors. To identify meaningful epigenetic markers of breast cancer risk, in Aim 1 we will assess differences in histone modifications between cell lines from low and high breast cancer risk contexts. These modifications will be investigated at the level of nuclear domains known to participate in replication and differentiation (e.g., telomeric and pericentromeric chromatin), and at genes important for epithelial homeostasis. Markers of interest will be validated on archival breast tissue biopsy sections of women at different breast cancer risk levels. In Aim 2, we will determine the architectural and epigenetic responses of the same cell models to dietary and chemically-based modulators of breast cancer risk in order to establish the functionality of the models. Throughout the project we will use a unique high-throughput (HTP) 3D culture method. Future developments would make use of the markers identified and the HTP 3D culture systems to decipher cancer initiation mechanisms and for large scale screening of compound repositories for potential preventive agents using emerging microscopy-based state-of-the-art technologies.